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Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 1106-1107
CORRESPONDENCE
No Evidence for MLL/AF4 Expression in Normal Cord Blood Samples
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LETTER |
To the Editor:
In a recent issue of Blood, Uckun et al1 reported a
high frequency of the MLL/AF4 expression not only among infant patients with acute lymphoblastic leukemia (ALL) but also among children older
than 1 year (12 out of 127) at the diagnosis of the disease. This group
with reverse transcription-polymerase chain reaction (RT-PCR)
positivity only in the second round comprises both patients with or
without cytogenetically detectable t(4;11) and with or without MLL gene
rearrangement detectable on the DNA level by the Southern blot
analysis. Moreover, investigators found low levels of MLL/AF4
expression also in a significant proportion of fetal tissues and in
bone marrow (BM) of 1 healthy infant out of 6 tested. Based on these
results, Uckun et al conclude that MLL/AF4 fusion transcripts can be
present in normal hematopoietic cells and that their presence per se is
not sufficient for leukemic transformation of normal lymphocyte
precursors. Furthermore, the authors question the reliability of RT-PCR
detection of minimal residual disease in MLL/AF4-positive patients as
well as its use for the screening of cord bloods before transplant. In
contrast, our results show that in pediatric ALL patients, nested
RT-PCR may achieve detection limit 0.01% without compromising
specificity. Moreover, our findings suggest that MLL/AF4 fusion is not
expressed in the cord blood samples of healthy newborns.
We conducted a large prospective study on pediatric ALL on behalf of
the Czech Pediatric Hematology Working Group, and analyzed the
frequency and clinical impact of various translocations, including t(4;11). We used the nested RT-PCR approach for the detection of the
fusion gene MLL/AF4. RNA was extracted using a method modified after
the single-step technique described by Chomczynski and
Sacchi.2 RNA integrity and quantity was evaluated in
nondenaturing agarose gel electrophoresis. The single-round
amplification of ABL exons A2 to A3 was then used for cDNA quality
control. As we know from our previous work (Divoky et al, manuscript
submitted), exon 6 of the MLL gene is frequently absent in
the alternatively spliced MLL/AF4 transcripts. Therefore, in contrast
to the method used by Uckun et al, our RT-PCR used primers in MLL exon
5 for both the first and second round of our PCR. The sensitivity of
this approach, evaluated using limiting dilution of the
MLL/AF4-positive cells into normal cells, is 10 4
(0.01%), the sensitivity identical to the one described by Uckun et al.
We started our prospective study in June 1994. Since then, we found 7 infant patients positive for MLL/AF4 out of 11 tested (64%),
contrasting with only 1 out of 206 patients aged 1 to 18 years (0.5%).
Out of these 8 children, 7 were positive in the first round and 1, tested only after the induction therapy block and at subsequent
relapse, in the second round. Cytogenetic analysis was available in 5 of these patients, and in 4 cases t(4;11) was detected. In 1 remaining
patient, cytogenetic analysis revealed deletion in 11q23 locus. To test
the hypothesis that MLL/AF4-expressing cells could be present among
hematopoietic cells of healthy newborns, we applied the identical
two-round RT-PCR approach to the cord blood samples from 103 full-term
healthy newborns born in our hospital from February to March 1998 and
found none of them to be MLL/AF4-positive.
The individuals positively tested for MLL/AF4 expression in the study
of Uckun et al could be divided (according to the results of
cytogenetic analysis, Southern blot for MLL rearrangement, and RT-PCR)
into 3 subgroups. The first subgroup (A) comprises patients with
t(4;11)+, MLL rearrangement present, and RT-PCR positivity in the first
round (standard PCR), thus representing expected results. Subgroup B
then includes t(4;11)+ or  , MLL rearrangement +, and RT-PCR second
round positives; and the subgroup C includes t(4;11), MLL
rearrangement , and RT-PCR second round positives. Patients
encompassed in the two latter subgroups (B and C) must represent two
rather distinctive MLL/AF4-positive populations: one relatively large
(at least 5% of all mononuclear cells, taking into account that
MLL-rearranged cells are detectable by Southern blot analysis, but
Southern blots presented in the report seem to indicate much higher
proportion of MLL-rearranged cells) with low expression of MLL/AF4, and
the other relatively minute population with high expression level
(subgroup C). Remarkably, without exception all examined normal
samples (comprising fetal livers, fetal BM samples, and BM
of presumably healthy infants) fall into the last subgroup, whereas
majority of ALL patients (noninfant patients in particular) fall under
the characteristics of the previous one. Although it is not clear from
the report of Uckun et al how many of the "normal"
MLL/AF4-positive samples were actually analyzed by Southern
blot, this distribution seems to be notable. However, the authors did
not attempt to interpret this distinction.
In the report of Uckun et al, we find another intriguing phenomenon. We
know from our previous experience (Divoky et al,
manuscript submitted) that leukemic cells of MLL/AF4-positive patients
express usually more than one (and up to six) different alternatively spliced transcripts, representing very specific pattern of PCR products
in the post-PCR gel. Surprisingly, gel photographs included in the
report of Uckun et al show only a very limited amount of alternatively
spliced variants (3 in total), with no more than one PCR product
visible in the gel for one individual patient. DNA-based
analysis, documenting distinctive patient-specific junction of
MLL and AF4 genetic material, would ultimately exclude the possibility
of PCR artifact or carry-over contamination.3 Southern blot
analysis shown by Uckun et al is used as an independent method to
support some of the RT-PCR-based findings. However, to cite an
instance, it is technically impossible to detect the same
rearrangement using the PCR with primers in exon 6 and by the Southern
blot with 98.40 probe after BamH1 restriction (Table
4, patients INF-6, PEDS-5). The probe and the restriction
sites lie upstream from the sixth exon.4,5
One of the conclusions made by Uckun et al was that nested RT-PCR may
not be suitable for MLL/AF4 screening of cord blood because they have
found positive samples among presumably healthy fetal or infant tissues
(other than cord blood). We consider analysis of healthy cord blood
more appropriate for such a statement.
We tried to summarize some intriguing details in the study of Uckun et
al that in our opinion need further elucidation. Our data do not
support any of the hypotheses implied by Uckun et al. We found neither
patients with low expression of MLL/AF4 fusion gene nor the
MLL/AF4-positive cells in any of 103 cord blood samples tested. We
believe that nested RT-PCR method can be used for the MRD follow-up in
t(4;11)+ patients as well as for potential screening of cord blood
before transplant. Uckun's data, although showing unanticipated
biological observation, should not be interpreted as an argument for
considering an MLL/AF4-positive specimen free of leukemic blasts.
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ACKNOWLEDGMENT |
This work was supported in part by Grants No. 3425-3 and 3920-3 from
Ministry of Health, Czech Republic.
J. Trka
J. Zuna
O. Hru ák
K. Michalová
K. Mu íková
M. Kalinová
J. Starý
Laboratory of Molecular Genetics
2nd Department of Paediatrics
Charles University
Prague
Prague, Czech Republic
| |
REFERENCES |
1.
Uckun FM, Herman-Hatten K, Crotty ML, Sensel MG, Sather HN, Tuel-Ahlgren L, Sarquis MB, Bostrom B, Nachman JB, Steinherz PG, Gaynon PS, Heerema N:
Clinical significance of MLL-AF4 fusion transcript expression in the absence of a cytogenetically detectable t(4;11)(q21;q23) chromosomal translocation.
Blood
92:810, 1998[Abstract/Free Full Text]
2.
Chomczynski P, Sacchi N:
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:156, 1987[Medline]
[Order article via Infotrieve]
3.
Gale KB, Ford AM, Repp R, Borkhardt A, Keller C, Eden OB, Greaves MF:
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94:13950, 1997[Abstract/Free Full Text]
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Chen CS, Sorensen PH, Domer PH, Reaman GH, Korsmeyer SJ, Heerema NA, Hammond GD, Kersey JH:
Molecular rearrangements on chromosome 11q23 predominate in infant acute lymphoblastic leukemia and are associated with specific biologic variables and poor outcome.
Blood
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Downing JR, Head DR, Raimondi SC, Carroll AJ, Curcio-Brint AM, Motroni TA, Hulshof MG, Pullen DJ, Domer PH:
The der(11)-encoded MLL/AF-4 fusion transcript is consistently detected in t(4;11)(q21;q23)-containing acute lymphoblastic leukemia.
Blood
83:330, 1994[Abstract/Free Full Text]
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